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Outline

Bio-sensors are invaluable tools to easily visualize and track changes in the cell and its environment. Thus they are important components of the Aspergillus arsenal, which is an array of parts and devices to make Aspergillus niger more amenable to work with. We envision the use of ATP and pH bio-sensors to monitor the micro-environment in the cell where a pathway of interest has been targeted, in our case, the lovastatin biosynthesis pathway.

Such bio-sensors can provide quantitative information that can prompt us to maybe direct pathways to proper cell compartments or change the growth conditions to increase the yields. The actual fluctuations of proton and ATP concentrations in live cells can be pursued corresponding to different growth conditions. Consequently, filling gaps in knowledge about the cell physiology which is also essential in the context of host engineering. For example, the ATP bio-sensor will be instrumental in exploring the metabolic capabilities of the single cell like phenotype of A. niger at 44'C, which has been a burning question for many years now.

Introduction

A great array of new and existing biological devices and circuits have been designed and streamlined for useful purposes. However, currently there is not enough emphasis on how these devices and circuits respond to changes in their physiological surroundings, such as the effect of changes in pH and the availability of ATP. Thus we tested bio-sensors that enable us to monitor the dynamic changes of pH and ATP in live cells. The ATP bio-sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET) and the pH bio-sensor is based on recombinant red fluorescence protein (pHRed). Furthermore, measurements using both bio-sensors are based on ratiometric methods, which eliminate the influence of photobleaching, laser intensity and etc. By combining multicolour, pHRed (red fluorescence) and FRET (green fluorescence), we can simultaneously image intracellular pH and ATP, as changes in metabolism and ATP availability often correlates with the changes in pH. These bio-sensors are useful to non-invasively quantify specific measurements in living cells.

pH biosensor

Regulation of intracellular pH is important for the metabolism of a functional cell as it is tightly regulated by the complex interactions between the consumption, production, transportation and buffering of H+ [3][4]. To maintain protein stability, enzyme and ion channel activity, growth and division, it is essential to retain pH within their physiological range of pH 7.5 [1]. In addition, it is important to maintain a proton gradient between the cytoplasm and mitochondria, which allow the formation of ATP to sustain their metabolism. Aspergillus niger is a GRAS organism that is among the favourites in producing organic acid as it can tolerate acidic environment. Since Aspergillus niger can produce high amount of organic acid, it would be interesting to investigate the fluctuation and difference in pH between the cytoplasm and mitochondria during the production of citric acid. This would be interesting to determine whether the pH, at a certain position in a filamentous fungi, is optimal for a certain reaction. This will allow us to direct and target a metabolic pathway into its appropriate compartments for optimal production. This will also allow us to elucidating the mechanism in how fast the homeostatic pH is being regulated when acid is production with extracellular acidic environment.

pHRed

A novel approach in measuring intracellular pH in a non-invasive way has been developed by Mathew Tantama et. al. 2011, by using a recombinant red fluorescent protein (RFP), termed mKeima, had been genetically engineered into a S213A mutant, called pHRed. This will allow it to exhibit dual excitation peaks at 440 nm and 585 nm and an emission peak at 610 nm that respond ratiometrically well to pH changes [5]. The advantages of using a ratiometric method is that the estimation of pH is not affected by photobleaching, changes in focus of the microscope, variations in laser intensity, optical path length and illumination intensity. The excitation peak at 585 nm is caused by the anionic chromophore in an acidic condition whereas the excitation peak at 440 nm is caused by the protonated neutral chromophore in an alkaline condition.

Figure 1) How different pH levels affect the excitation peaks of pHRedA. A: pHRed in acidic condition has an excitation peak at 585 nm with an emission peak at 610 nm. B: pHRed in alkaline condition has an excitation peak at 440 nm with an emission peak 610 nm.

The pH can be calculated by measuring the fluorescence intensity ratio between F585 and F440 (F585/ F440); and compare it with a calibration curve, see FIG. pH responses to ratio intensity of F585/ F440 are not affected in different buffer concentration such as Na+, K+, Mg2+, Cl−, Ca2+, HCO3−, H2O2, DTT, and temperature (21°C to 37°C). Based on these findings, pHRed had proven to be useful in monitoring and analyzing the dynamic changes in pH.

Figure 1)[5]

FRET sensor

Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.

ATP FRET sensor (Imamura et al. 2009)

FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.

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Strategy and Approach

To utilize pHRed for in vivo measurements of pH inside a filamentous fungi during citric acid production, two constructs were made. With one targeting into the cytoplasm and the other targeting into the mitochondrial by transforming fungi with a naked pHRed sensor and pHRed sensor fused with mitochondrial signal peptide, respectively. The mitochondrial signal peptide has been proven to target into the mitochondria of Asp. Niger [2] . To measure pH, a calibration curve is established by purifying streptagII fused at N-terminus of pHRed proteins from sonificated E. coli, and is observed under a fluorescence microscope.

Research Aims

1.Make 2 contructs for targeting pHRed to cytoplasmic and mitochondrial compartments.
2.Purify strepTagII-pHRed from sonificated E. coli. and make a calibration curve.
3.Transform Aspergilllus niger with cytoplasmic pHRed and mitochondrial pHRed
4.Measure in vivo pH during citric acid production by Asp. Niger in time lapse using a fluorescence microscope

Results

After transformation of DH5α with 1µl of mixture (gibson assembly with pJet1.2 blunt vector), 7 colonies were picked and were cultured in 5 ml LB medium with ampicillin. Afterwards, plasmids were isolated using a miniprep kit (thermo scientific) according to their protocol using a homemade silicon column. Culture #1 till #3 were digested with pstI and notI; culture #4 till #7 were digested with nsiI and notI, see Figure 1.

Figure 1) lane 1, 3 and 4 contain pJet1.2 with the appropriate mit-pHRed (803 bp). Lane 5-7 contain the pJet1.2 with the appropriate pHRed (725 bp).

References

1.Bagar, T., K. Altenbach, et al. (2009). "Live-Cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe." Eukaryot Cell 8(5): 703-712.
2.Blumhoff, M. L., M. G. Steiger, et al. (2013). "Targeting enzymes to the right compartment: Metabolic engineering for itaconic acid production by Aspergillus niger." Metabolic Engineering 19(0): 26-32.
3.Felle, H. H. (1996). "Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs." Journal of Experimental Botany 47(7): 967-973.
4.Madshus, I. H. (1988). "Regulation of intracellular pH in eukaryotic cells." Biochem J 250(1): 1-8.
5.Tantama, M., Y. P. Hung, et al. (2011). "Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor." J Am Chem Soc 133(26): 10034-10037.